To understand the mechanisms of combinatorial control by transcription factors (Spitz et al., 2012), it was reasoned that an important contribution would be the ability to precisely map transcription factor binding footprints in vivo at a single nucleotide resolution. The occupancy of specific transcription factors can be mapped by chromatin immunoprecipitation (ChIP) coupled to deep sequencing (ChIP-seq), but the resolution of this technique is limited since a minimal DNA fragment size is required for unique alignment to the genome (Bardet et al., 2013). In an improvement to ChIP-seq called ChIP-exo, the immunoprecipitated chromatin fragments are digested by lambda exonuclease, which digests one strand of the double stranded DNA (dsDNA) in a 5′-to-3′ direction and stops when it encounters a cross-linked protein. In this manner, the exact bases bordering a DNA-bound protein (the ‘stop bases’) may be accurately and uniquely mapped, revealing the binding footprint of a protein at essentially nucleotide resolution.
ChIP-exo has proven to be a very powerful technique and has revealed interesting insights into the binding of transcription factors and chromatin remodeling factors (Venters et al., 2013, Rhee et al., 2012a, Rhee et al., 2012b). However, there are technical hurdles in establishing and applying the technique to biological problems. Notably, the additional wash and digestion steps in ChIP-exo produce lower amounts of DNA as compared to conventional ChIP-seq experiments. The amount of recovered DNA fragments, however, is critical for the quality of a ChIP library. For amplification during library preparation, DNA fragments have to successfully complete two inefficient ligation steps in order to acquire adaptors on both of their ends (Rhee et al., 2012b, Rhee et al., 2011). Low amounts of starting DNA often lead to over-amplification artifacts during PCR, producing noisy data that are not reproducible (Kivioja et al., 2012, Casbon et al., 2011). The original ChIP-exo protocol was designed for a particular platform—the SOLiD platform—it would be beneficial to have a protocol that works with other platforms, such as, e.g., Illumina-based platforms. (See, e.g., Venters et al., 2013, Serandour et al., 2013). The present invention is directed to meeting the above-identified and other needs and that has high robustness and reproducibility, even with transcription factors whose ChIP experiments typically yield low amounts of DNA.